Immunochemistry (IHC) is a common tool in medical diagnostics and it is also usual for the assessment of therapeutic biomarkers. The latter, in particular, often require a quantitative evaluation of the extent of their presence. The application of antibodies to cells and tissues presents specific difficulties beyond those encountered when these reagents are applied to purified proteins immobilized onto solid supports in or solution. There are many factors that can affect immunodetection, among these fixation and preparation of tissue, duration and type of antigen retrieval and antibody specificity. An additional difficulty is the ability to detect targets present at low levels. In common with soluble assays, this becomes a matter of increasing signal without raising the level of nonspecific background. The approach that has been most commonly explored is signal amplification, which is achieved by successive rounds of enzymatic reactions.
3,3′-Diaminobenzidine (DAB) is a chromogenic substrate of horse radish peroxidase (HRP) that is widely used for visualizing of target proteins in histological samples which are labeled with peroxidase activity. The method utilizes HRP linked to antibodies targeted to proteins of a sample that deposits DAB from a solution to the sites of targeted proteins and thereby labels the proteins. The method is not especially sensitive and therefore suitable for detection of relatively abundant target proteins. The signal associated with DAB deposits cannot be further amplified. Other drawbacks to mention are that the method demands rather high amounts of target specific antibodies to saturate all target sites and it is relatively time consuming. Furthermore, the method provides a uniform staining pattern that appears to the microscopist as a homogeneous color with intracellular resolution of cellular structures, e.g. membrane, cytoplasm, and nucleus, which makes it impossible to quantify the staining accurately.
Recently, a novel HRP-DAB-based IHC visualization system has been described. This system utilizes DAB not only as a chromogenic substrate of HRP to label targets, but also as an agent which cross-links other detectable HRP substrates in aqueous solutions with the assistance of HRP and deposits them thereby in the vicinity of the immobilized HRP (see WO2009036760, WO2010094283 and WO2010094284). In histological samples the method produces a staining pattern that is similar to a traditional HRP-DAB staining, but, compared to the traditional staining, this staining is much more target specific and sensitive and the procedure is much faster and robust.
The new method does not allow direct approximating the quantity of the target to the quantity of the stain in a sample, because the correlation between these two quantities is not linear. Accordingly, the quantity of a target in a histological sample visualized by all these methods can only be assessed relatively, not precisely. However, under certain conditions, this newly described HRP-DAB target labeling system is capable of amplifying a signal associated with a single target so strongly, that that single targets, such as single protein or nucleic acid molecules, may be visualized in the samples individually as large dots of color or fluorescence and detected by using ordinary low magnification bright-field or fluorescence optics (see WO2011047680). The visually distinct single dots with a diameter up to 4 microns may be then easily manually or automatically quantified in the sample and the amount of the target may be determined very precisely (see PCT/DK 2011/000131).